Current psychiatric pharmacotherapy options leave much to be desired, and manipulation of N-methyl-D-aspartate receptors (NMDARs) by positive and negative regulators has therapeutic benefit in major psychiatric disorders including schizophrenia and depression. We propose to capitalize on our recent discovery of a novel, very potent, and potentially endogenous positive modulator of NMDARs. The prototype is the major cholesterol metabolite in brain, 24S-hydroxycholesterol (24OH). Oxysterols like 24OH are cellular lipids generated from oxidation of cholesterol. Prevailing views suggest 24OH is synthesized as a convenient waste disposal vehicle for the neuronal pool of cholesterol that turns over in brain, but we have discovered that 24OH modulates NMDAR function at concentrations well below those measured in brain. We will leverage innovative chemical biology and physiological approaches toward understanding the mechanisms of 24OH. Our preliminary data suggest that 24OH and a synthetic analogue, Org-1, have unusual potentiating actions at NMDARs that are antagonized by another cholesterol metabolite, 25-hydroxycholesterol. We hypothesize that 24OH has direct effects on NMDAR channel gating through a novel receptor binding site. To understand mechanisms of oxysterol neuromodulation, we exploit a longstanding interdisciplinary collaboration with a synthetic chemistry laboratory, directed by Douglas Covey. We propose to use novel chemical tagging strategies to create analogues for cell biological and physiological studies. These approaches will yield insight into subcellular domains of exogenous oxysterol actions and insight into the peculiar kinetics of oxysterol action. We will also investigate the effects of 24OH and Org-1 on synaptic plasticity and behavior in wild type mice and in mice deficient in the enzyme that produces 24OH. Preliminary data suggest that 24OH and Org-1 enhance synaptic plasticity and cognition. This work will be complemented by measurements of endogenous 24OH and Org-1 levels under varied experimental conditions by collaborators at Weill Cornell Medical College. We have a strong interdisciplinary track record that will foster rapid progress. Upon completion of these studies, we expect to have elucidated mechanisms and effects of a novel neuromodulator with broad importance.